Plasmin-resistant streptokinase

Abstract

The invention features modified streptokinase (SK) molecules which are resistant to plasmin cleavage including a recombinant fusion protein in which the amino terminus of SK was blocked with a peptide, a recombinant fusion protein in which an amino-terminal deleted SK was blocked with a peptide, and a mutated SK in which plasmin-cleavage sites were altered to render those sites resistant to enzymatic cleavage.

What is claimed is: 
     
       1. A plasminogen-binding fragment of streptokinase, wherein (a) said fragment lacks 1 to 24 amino-terminal amino acids;   (b) said fragment is catalytically active; and   (c) the rate of in vitro degradation of said fragment in the presence of human plasminogen is at least 2 times slower compared to the rate of degradation of native streptokinase in the presence of human plasminogen, wherein the rate of degradation is measured by the appearance of plasmin cleavage products as detected by immunoblotting using anti-streptokinase antibodies, and   (d) wherein said fragment further comprises at least one mutation in a potential plasmin cleavage site which renders said cleavage site resistant to cleavage by plasmin.   
     
     
       2. The fragment of claim 1, wherein said fragment comprises the amino acid sequence of (SEQ ID NO:4). 
     
     
       3. A polypeptide comprising a plasminogen-binding fragment of streptokinase, wherein (a) said fragment is catalytically active; and   (b) the rate of in vitro degradation of said polypeptide in the presence of human plasminogen is at least two times slower compared to the rate of degradation of native streptokinase in the presence of human plasminogen, wherein the rate of degradation is measured by the appearance of plasmin cleavage products as detected by immunoblotting using anti-streptokinase antibodies, and   (c) wherein said polypeptide further comprises at least one mutation in a potential plasmin cleavage site which renders said cleavage site resistant to cleavage by plasmin.   
     
     
       4. The polypeptide of claim 3, wherein said mutation is selected from the group consisting of R10A, K36A, R45A, K51A, K59A, K61A, K147A, K333, R232A, K257A, K298A, K309A, R234A, R363A, K386A, K372A, R388A, R394A, and R401A. 
     
     
       5. The polypeptide of claim 4, wherein said polypeptide comprises R10A, K36A, R45A, K51A and K59A (SEQ ID NO:17). 
     
     
       6. The polypeptide of claim 4, wherein said polypeptide comprises R10A, K36A, R45A, K51A, K59A and K386A (SEQ ID NO:18). 
     
     
       7. A compound comprising (a) a plasminogen-binding fragment of streptokinase and (b) a blocking group at the amino-terminus of said fragment, wherein (i) said compound is catalytically active; and   (ii) the rate of in vitro degradation of said compound in the presence of human plasminogen is at least 2 times slower compared to the rate of degradation of native streptokinase in the presence of human plasminogen, wherein the rate of degradation is measured by the appearance of plasmin cleavage products as detected by immunoblotting using anti-streptokinase antibodies, and   (iii) said blocking group is a non-peptide blocking group.   
     
     
       8. The compound of claim 7, wherein said blocking group is attached to the fragment by glycosylation or myristolization.